Left-Right Side-Specific Neuropeptide Mechanism Mediates Contralateral Responses to a Unilateral Brain Injury

An experimental design. Rats received UBI or sham surgery on day 0. HL-PA was analyzed before and after treatment with opioid antagonists or saline; and before and after complete spinal cord transection. In design 1, a time course of HL-PA after the UBI or sham surgery was analyzed. In design 2, naloxone, NTI, or saline were injected to the left-side and right-side UBI rats. HL-PA was evaluated on day 1, and on the test day before and 30 min after the injection. In design 3, effects of nor-BNI and β-FNA on HL-PA induced by the left-side or right-side UBI were analyzed; the antagonists were administered 1 d before the analysis. In design 4, effects of spinal cord transection, and effects of naloxone in rats with transected spinal cord on the HL-PA were examined.

The HL-PA induced by UBI. Effects of the general opioid antagonist naloxone. HL-PA was analyzed in the UBI or sham surgery rats on days 1 and 3; and on day 3 in UBI rats 30 min after treatment of with naloxone or saline. The number of rats is shown on the plots (design 2; naloxone: 14 left and 3 right UBI rats; saline: 5 left and 5 right UBI rats). HL-PA was measured in millimeters as the length of the projection of the line connecting symmetric hindlimb distal points on the longitudinal axis of the rat and presented as the MPA. A, The MPA is plotted as median, 95% HPDCI, and posterior distribution from Bayesian regression. B, Contrasts in MPA between rat groups are plotted as medians, 95% HPDCI and densities from Bayesian sampler. Adjusted p values are shown if they are <0.05 for differences in MPA between rat groups. Differences were considered to be significant if 95% HPDCI did not include zero, and adjusted p values were <0.05. Time course, comparison of isoflurane and pentobarbital anesthesia, and effects of spinal cord transection and administration of saline or naloxone on HL-PA after the left and right-side UBI are shown on Extended Data Figure 2-1.

Effects of naloxone on the left-right asymmetry index for the spike number (AI_SN) of the EDL, Int, and PL muscles in the UBI rats. A, Experimental design. Rats with the right UBI or right sham surgery (Sh) were exposed to complete spinal cord transection and decerebration, and were infused with naloxone or infusion buffer. The NWRs of the left and right limbs were stimulated and recorded ipsilaterally. EMG activity of left and right hindlimb muscles was evoked by electrical stimulation of symmetrical digits of left and right hindlimbs. Four groups consisted of 39 rats including 11 sham and 14 UBI not treated (control) rats, and seven sham and seven UBI rats treated with naloxone rats were analyzed between third and sixth day after UBI or sham surgery. The number of animals which data were used in statistical analysis is shown in Extended Data Figure 3-1. B, C, Representative EMG responses of the Int muscle to electrical stimulation of digit 4 of the UBI rats. D, The left-right asymmetry index for the spike number (AI_SN = log₂[(1 + SN_Left)/(1 + SN_Right)]; where Left and Right were values for the left and right side). E, Difference between the naloxone-treated and naloxone-untreated (control) groups in AI_SN [ΔAI = (AI_Naloxone – AI_Control)]. F, Difference between the UBI and respective sham surgery groups in AI_SN [ΔAI = (AI_UBI – AI_sham)]. Medians, 95% HPDC intervals and densities of posterior estimates from Bayesian sampler are plotted. Asymmetry and contrast between the groups were defined as significant if 95% HPDC intervals for AI_SN did not include zero, and adjusted p values were <0.05.

Lateralized expression of the opioid genes and opioid peptides in the lumbar spinal cord. Data for five animal groups were combined separately for each the left and right lumbar spinal halves (n = 50), Log₂-scaled and compared using Wilcoxon matched pairs test. Data are shown for the expression levels of the μ-opioid (Oprm1), δ-opioid (Oprd1), and κ-opioid (Oprk1) receptor genes; the prodynorphin (Pdyn) and proenkephalin (Penk) opioid peptide precursor genes; and for the Oprk1/Oprd1, Oprk1/Oprm1, Oprd1/Oprm1, and Pdyn/Penk ratio, along with data for opioid peptides LER and MEAP, the Pdyn and Penk markers, respectively, and for the LER/MEAP ratio. White and dark gray boxes denote the left and right spinal cord. The horizontal line in the box represents the median; the box hinges represent the first (Q1) and third (Q3) quartiles. Upper and lower whiskers extend from the hinge to the highest/lowest value that lies within the 1.5 interquartile range (IQR) of the hinge. The Bonferroni adjusted p values are shown. Analysis of replication sample is shown on Extended Data Figure 4-1.

Effects of UBI on intra-area correlations between the opioid and neuroplasticity-related genes (genes analyzed are shown in Extended Data Fig. 5-1) in the left and right halves of the spinal cord. Heatmap for intra-area Spearman’s rank correlations between expression levels of five opioid system genes and 13 neuroplasticity-related genes in the left and right spinal cord for the naive group, the left and right sham surgery groups, and left and right UBI groups (five groups; n = 10 rats/group). Significance of differences in the proportion of positive and negative correlations among five groups, and, separately between two animal groups, were assessed by the Fisher’s exact test with 2 × 5 or 2 × 2 contingency tables, respectively. Two-tailed uncorrected p value is shown. Differences among five animal groups for the left and right spinal halves: p = 0.048 and p = 8.4e-05, respectively. Differences for the right spinal half between the left UBI versus left sham surgery: p = 2.4e-04; and between the right UBI versus right sham surgery: p = 0.043.

Effects of nor-BNI, β-FNA, and NTI, the selective κ-opioid, μ-opioid, and δ-opioid antagonists, respectively, on HL-PA induced by the left and right UBI. The UBI rats were treated with nor-BNI (BNI) or β-FNA (FNA) 1 d before (design 3), or with NTI 30 min before (design 2) HL-PA analysis. A, The MPA is plotted as median, 95% HPDCI, and posterior distribution from Bayesian regression. The number of rats is shown on the plot. B, Contrasts in MPA between a rat group treated with a given antagonist and respective saline group (Ctrl). C, Contrasts in MPA between rat groups with the left or right-side UBI; the UBI rats in each pair of groups were treated with the same antagonist or saline (Ctrl). Contrasts are plotted as medians, 95% HPDCI and densities from Bayesian sampler. Adjusted p values are shown if they are <0.05 for differences in MPA between the groups. Differences were considered to be significant if 95% HPDCI did not include zero, and adjusted p values were <0.05.